Chemical labeling strategies for cell biology

Methods to visualize, track, measure and perturb proteins in living cells are central to biomedicine's efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have revolutionized such studies, they have shortcomings, which have spurred the creation of alternative approaches to chemically label proteins in live cells. In this review we highlight research questions that can be addressed using site-specific chemical labeling and present a comparison of the various labeling techniques that have been developed. We also provide a 'roadmap' for selection of appropriate labeling techniques(s) and outline generalized strategies to validate and troubleshoot chemical labeling experiments.

[1]  H. Vogel,et al.  Reversible site-selective labeling of membrane proteins in live cells , 2004, Nature Biotechnology.

[2]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Verkman,et al.  Receptor-mediated Targeting of Fluorescent Probes in Living Cells* , 1999, The Journal of Biological Chemistry.

[4]  R. N. Trelease,et al.  Overexpression and Mislocalization of a Tail‐Anchored GFP Redefines the Identity of Peroxisomal ER , 2003, Traffic.

[5]  M. Sheetz,et al.  Methotrexate conjugates: a molecular in vivo protein tag. , 2004, Angewandte Chemie.

[6]  N. Johnsson,et al.  Multicolor imaging of cell surface proteins. , 2005, Journal of the American Chemical Society.

[7]  A. Ting,et al.  Site-specific labeling of proteins with small molecules in live cells. , 2005, Current opinion in biotechnology.

[8]  M. Howarth,et al.  Targeting quantum dots to surface proteins in living cells with biotin ligase. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Sheetz,et al.  In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag , 2005, Nature Methods.

[10]  Mark H Ellisman,et al.  A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells , 2005, Nature Methods.

[11]  R Y Tsien,et al.  Specific covalent labeling of recombinant protein molecules inside live cells. , 1998, Science.

[12]  H. Vogel,et al.  A general method for the covalent labeling of fusion proteins with small molecules in vivo , 2003, Nature Biotechnology.

[13]  Brent R. Martin,et al.  Mammalian cell–based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity , 2005, Nature Biotechnology.

[14]  G. Nolan,et al.  Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores. , 1998, Chemistry & biology.

[15]  A. Juillerat,et al.  Engineering Substrate Specificity of O6‐Alkylguanine‐DNA Alkyltransferase for Specific Protein Labeling in Living Cells , 2005, Chembiochem : a European journal of chemical biology.

[16]  N. Johnsson,et al.  Specific labeling of cell surface proteins with chemically diverse compounds. , 2004, Journal of the American Chemical Society.

[17]  Hideo Tanaka,et al.  Multiphoton excitation–evoked chromophore-assisted laser inactivation using green fluorescent protein , 2005, Nature Methods.

[18]  G. Davis,et al.  Transgenically Encoded Protein Photoinactivation (FlAsH-FALI) Acute Inactivation of Synaptotagmin I , 2002, Neuron.

[19]  G. Nolan,et al.  A general approach for chemical labeling and rapid, spatially controlled protein inactivation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  G. Nolan,et al.  In vivo targeting of organic calcium sensors via genetically selected peptides. , 2004, Chemistry & biology.

[21]  F. Marshall,et al.  In vivo molecular and cellular imaging with quantum dots. , 2005, Current opinion in biotechnology.

[22]  Roger Y Tsien,et al.  Imagining imaging's future. , 2003, Nature reviews. Molecular cell biology.

[23]  Ken Jacobson,et al.  Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins , 2002, Nature Cell Biology.

[24]  J. Lippincott-Schwartz,et al.  Development and Use of Fluorescent Protein Markers in Living Cells , 2003, Science.

[25]  D. Holt,et al.  A versatile synthetic dimerizer for the regulation of protein-protein interactions. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[26]  H. Vogel,et al.  Labeling of fusion proteins with synthetic fluorophores in live cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Roger Y Tsien,et al.  Genetically targeted chromophore-assisted light inactivation , 2003, Nature Biotechnology.

[28]  Robert E Campbell,et al.  New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. , 2002, Journal of the American Chemical Society.

[29]  A. Plückthun,et al.  Stability engineering of antibody single-chain Fv fragments. , 2001, Journal of molecular biology.

[30]  M. Howarth,et al.  Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase , 2005, Nature Methods.

[31]  Thomas J Deerinck,et al.  Multicolor and Electron Microscopic Imaging of Connexin Trafficking , 2002, Science.

[32]  Catherine Proenza,et al.  The protein-labeling reagent FLASH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins , 2001, Pflügers Archiv.

[33]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[34]  R. Tsien.  Fluorescent indicators of ion concentrations. , 1989, Methods in cell biology.

[35]  N. Allbritton,et al.  Source of nuclear calcium signals. , 1994, Proceedings of the National Academy of Sciences of the United States of America.